Anchorage-dependent cells, especially diploid anchorage-dependent cells are used in a wide range of processes: for the production of health care products such as vaccines and recombinant proteins in large-scale bioprocesses, for the generation of artificial tissues used in the treatment of human injuries, for experimental investigations, for in vitro toxicology, for screening and testing of new drugs, etc.
Conventionally, anchorage-dependent cells are cultured in media containing serum or other animal-origin components as substitutes for the serum, such as bovine serum albumin (BSA) or protein hydrolysates. Serum or animal-origin components are also used during cell subcultivation and in cell cryopreservation. Serum is a major source for metabolites, hormones, vitamins, iron (transferrin), transport proteins, attachment factors (e.g. fibronectin), spreading and growth factors. It is required for the growth of many animal cells culture in vitro. In addition, serum acts as buffer against a variety of perturbation and toxic effects such as pH change, presence of heavy metal ions, proteolytic activity, or endotoxins. Albumin is the major protein component of serum and exerts several effects which contribute to the growth and maintenance of cells in culture: it acts as a carrier protein for a range of small molecules and as a transporter for fatty acids which are essential for cells but are toxic in the unbound form.
Diploid anchorage-dependent cells are routinely grown on plastic surface, glass surface or microcarriers. The cells attach and spread out by attachment factors such as fibronectin (F. Grinnel & M. K. Feld Cell, 1979, 17, 117-129). Trypsin is one of the most common animal-derived component used for cell detachment during cell passaging (M. Schröder & P. Friedl, Methods in Cell Science, 1997, 19, 137-147; O. W. Mertens, Dev Biol Stand., 1999, Vol 99, pp 167-180). It must be inhibited by serum or soybean trypsin inhibitor after cell detachment in order to avoid cell damages. After detachment, cells are seeded at low density on a new surface where they can multiply and form a confluent cell layer before the next subcultivation. The purpose of passaging adherent cells is to multiply and obtain a sufficient amount of cells to carry out the aforementioned processes.
There are various disadvantages linked to the use of serum and of animal-derived components in these processes, mainly their cost, the batch to batch variability in their composition, their association with a higher contamination risk by adventitious agents, and the subsequent difficulties encountered in downstream processing (e.g. purification to get rid of the serum-proteins or of the introduced animal-derived proteins). Furthermore, as said above, it is reported that serum-free media are not suitable for anchorage dependent diploïd cells (O. W. Mertens, Dev Biol Stand., 1999, Vol 99, pp 167-180; O. W. Merten, Dev. Biol. 2002, 101,233-257).
A number of low-serum or serum-free medium formulations have been developed for anchorage-dependent cell culture, in particular for diploid anchorage-dependent cell culture (M. Kan & I. Yamane, Journal of Cellular Physiology, 1982, 111, 155-162; S. P. Forestell et al. Biotechnology and Bioenineering, 1992, Vol 40, pp 1039-1044). Attempts made with such media have not been satisfactory, mainly because diploid anchorage-dependent cell, which are not transformed, would need rather complex serum-free media supplemented with several growth factors and hormones, and also because production processes generally for such cells make use of serum at least during the biomass production phase (O. W. Merten, Dev. Biol. 2002, 101, 233-257). Furthermore, these media still contain components of animal origin, like BSA, protein hydrolysates, growth factors, transport proteins, amino acids, vitamins, etc. Very few attempts have been made to develop media formulations for anchorage-dependent cells which are totally free of components of animal origin. Formulations which are mostly animal-free are reported not to be able to sustain a cell growth rate equivalent to what is observed with serum and to only allow a few subcultivation steps before an early senescence is observed (B. J. Walthall & R. Ham Experimental Cell Research (1981) 134 303-311). Furthermore, primary cell cultures from anchorage-dependent cells almost always involve disaggregation of cell layers or tissue using a protease, mainly a serine-protease, of animal origin, thereby involving a risk of contaminating the cell culture with adventitious virus and causing unacceptable variability in cell growth due to batch to batch variation in the enzymatic activity of the protease. For example, the use of porcine/bovine trypsin in passaging anchorage-dependent cell cultures is a well-known technique (O. W. Mertens, Cytotechnology, 2000, 34, 181-183).
There exists a need therefore, in the field of diploid anchorage-dependent cell culture, to develop a cell culture medium which is substantially free from, preferably totally devoid of, animal-derived components, and is suitable for carrying a process for diploid anchorage-dependent cell culture with equivalent performances to that of a basal medium for the cell type supplemented with an appropriate serum, in terms of, for example, cell growth rate, senescence, cell morphology, viral or protein production, to these obtained with serum-containing processes.